Stem cells, in particular pluripotent stem cells such as ES cells and iPS cells, are receiving worldwide attention for their potential application to regenerative medicine. The culture and maintenance of stem cells without loss of their pluripotency usually requires the presence of feeder cells in the culture system, and as such feeder cells, mouse embryonic fibroblasts (MEFs) whose division has been arrested by radiation or antibiotic treatment are used. As the MEFs, STO cells etc. are usually often used, and SNL cells (McMahon, A. P. & Bradley, A. Cell 62, 1073-1085 (1990)) etc. are often used for generation of iPS cells. However, the use of feeder cells is a great restriction on clinical application of stem cells. In order to solve this problem, various cell adhesion molecules in place of feeder cells have been tried out as an extracellular matrix for stem cells. Among these, crude extracts prepared from mouse EHS sarcoma known to excessively produce basement membrane components (commercially available under the trade name of Matrigel) are reportedly highly effective for maintenance of the pluripotency of stem cells. Matrigel is known to abundantly contain basement membrane components such as laminin and type IV collagen, but the complete composition of Matrigel is still unknown. Further, Matrigel is a mouse derived product, and thus is not suitable for human stem cell culture in regenerative medicine.
For application of human stem cells to regenerative medicine, a feeder-free (no feeder cells are used) and xeno-free (the culture system contains no xenogeneic components) culture environment is desirable. Therefore, as an extracellular matrix of human origin, human vitronectin and human fibronectin have been tried out, but as compared with Matrigel, they are neither superior in maintenance of undifferentiated state of human stem cells or in adhesion efficiency thereof, nor satisfactory in terms of quality, source material availability, safety, etc. Thus, there is much to be improved in the development of extracellular matrices suitable for maintenance culture of human stem cells to be applied to regenerative medicine, and the development of new human-stem-cell culture techniques using human extracellular matrices having a uniform chemical composition is strongly desired.
Laminin is a major cell-adhesion molecule present in the basement membrane. Laminin is a large heterotrimeric glycoprotein consisting of three subunits termed α, β and γ chains, and has a molecular weight of 800,000 Da. The three subunits associate with each other through their coiled-coil domains in the C-terminal regions and assemble into a heterotrimer that is stabilized by disulfide bonds therein. The present inventors reported that recombinant human laminins (particularly, laminin 332, which consists of α3, β3 and γ2 chains, and laminin 511, which consists of α65, β1 and γ1 chains) are effective for maintaining the pluripotency of human ES cells (see Non Patent Literature 1). However, on the surfaces of human stem cells, adhesion receptors (membrane-bound molecules that are found on cell surfaces and mediate cell adhesion to extracellular matrices) other than laminin receptors are also expressed, and laminin molecules alone cannot use these adhesion receptors effectively. Then, strongly desired is the development of novel human extracellular matrices that have as natural an adhesion activity as possible and enable human stem cell culture in feeder-free conditions.